A Multicomponent Mannich Reaction Catalyzed by Hydrolases Immobilized on Titanate Nanotubes.

This study presents an innovative method for synthesizing ß-amino carbonylated compounds, specifically 2-[phenyl(phenylamino)methyl] cyclohexanone, achieving high conversions and diastereomeric ratios. Using trypsin or α-chymotrypsin in both free and immobilized forms on titanate nanotubes (NtsTi), synthesized through alkaline hydrothermal methods, successful immobilization yields were attained. Notably, α-chymotrypsin, when free, displayed a diastereoselective synthesis of the anti-isomer with 97 % conversion and 16 : 84 (syn : anti) diastereomeric ratio, which slightly decreased upon immobilization on NtsTi. Trypsin, in its free form, exhibited diastereoselective recognition of the syn-isomer, while immobilization on NtsTi (trypsin/NtsTi) led to an inversion of diastereomeric ratio. Both trypsin/NtsTi and α-chymotrypsin/NtsTi demonstrated significant catalytic efficiency over five cycles. In conclusion, NtsTi serves as an effective support for trypsin and α-chymotrypsin immobilization, presenting promising prospects for diastereoselective synthesis and potential industrial applications. Furthermore, it offers promising prospects for the diastereoselective synthesis of 2-[phenyl(phenylamino)methyl] cyclohexanone through multicomponent Mannich reaction and future industrial application.

Immobilization of the metagenomic lipase, LipG9, on porous pellets of poly-hydroxybutyrate produced by the double emulsion solvent evaporation technique.

This work aimed to produce porous poly-hydroxybutyrate (PHB) pellets in order to evaluate the pellets as a support for immobilization of the metagenomic lipase, LipG9. Four types of pelletized PHB particles with different morphological characteristics were obtained using the double emulsion and solvent evaporation technique (DESE). The micropores of these PHB pellets had similar average diameters (about 3 nm), but the pellets had different specific surface areas: 11.7 m2 g-1 for the PHB powder, 8.4 m2  g-1 for the control pellets (Ø < 0.5 mm, produced without the pore forming agent), 10.0 m2  g-1 for the small pellets (Ø < 0.5 mm), 9.5 m2  g-1 for the medium pellets (0.5 < Ø < 0.8 mm) and 8.4 m2  g-1 for the large pellets (Ø > 1.4 mm). Purified LipG9 was immobilized by adsorption on these pellets, and the results were compared with those obtained with PHB powder. The highest immobilization yield (83%) was obtained for the medium PHB pellets, followed by large (76%) and small (55%) PHB pellets. The activity of LipG9 immobilized on the pellets, for the synthesis of ethyl oleate in n-hexane, was highest for the medium pellets (22 U g-1 ). The immobilization yield was high for PHB powder (99%) but the esterification activity was slightly lower (20 U g-1 ). These results show that pelletized PHB beads can be used for the immobilization of lipases, with the advantage that pelletized PHB will perform better than PHB powder in large-scale enzyme bioreactors.

Transesterification of castor oil catalyzed by a fermented solid produced by Burkholderia contaminans using a bioreactor coupled with ultrasound irradiation.

In this work, ultrasound was used to assist the ethanolysis of castor oil in a solvent-free system, catalyzed by a dry fermented solid containing the lipase from Burkholderia contaminans (BCFS). Reactions were done at 45°C. The maximum conversion in Erlenmeyer flasks was 71% in 96 h, using a loading of 9% (mass of BCFS in relation to the mass of triacylglycerols in the castor oil) and a molar ratio of ethanol:oil of 6:1, with addition of ethanol in 12 steps. In a packed-bed reactor containing 12 g of BCFS, the conversions were 78% in 48 h, and 83% in 72 h with an ethanol to oil molar ratio of 3:1 and treatment with an ultrasound probe, with maximum power of 500 W, frequency of 20 kHz, and 75% of the maximum power. These results are promising given that, with an ultrasound assisted bioreactor, a higher conversion in a shorter time was achieved, with a lower ethanol to oil molar ratio than was the case in the Erlenmeyer flasks without ultrasound.

Biocatalytic asymmetric synthesis of secondary allylic alcohols using Burkholderia cepacia lipase immobilized on multiwalled carbon nanotubes.

The lipase from Burkholderia cepacia (BCL) was immobilized through physical adsorption on pristine and functionalized multiwalled carbon nanotubes (MWCNTs) with carboxyl or amine groups and used in the stereoselective acylation of (R,S)-1-octen-3-ol (1) and (R,S)-(E)-4-phenyl-3-buten-2-ol (4) with vinyl acetate. All immobilized preparations produced better results than free BCL. For (R,S)-4, 50% conversion and E > 200 were obtained in n-hexane or in solvent-free medium. For (R,S)-1, in solvent-free medium, the conversion was 38% with a slight increase in the E-value (E = 10).

Production of a fermented solid containing lipases from Penicillium roqueforti ATCC 10110 and its direct employment in organic medium in ethyl oleate synthesis.

The production and direct employment in organic medium in the ethyl-oleate synthesis of a fermented solid (FS) containing lipases by Penicillium roqueforti ATCC 10110 (PR10110) was investigated. For the production of this FS, the solid-state fermentation of different agroindustrial waste was used, such as: cocoa shell, sugarcane bagasse, sugarcane bagasse with cocoa shell, and cocoa shell with soybean oil and nutrient solution. The response surface methodology was used to study the effect of independent variables of initial moisture content and inductor concentration, as carbon source and inducer on lipase production. The characterization of the fermented solid in organic medium was also carried out. The highest lipase activity (53 ± 5 U g-1 ) was 16% higher than that obtained with the nonoptimized conditions. The characterization studies observed high stability of the FS in organic solvents for 5 h at 30°C, as well as at different temperatures, and the residual activity was measured against triolein. The FS was also able to catalyze ethyl-oleate synthesis maintaining high relative conversion over five reaction cycles of 96 h at 40°C in n-heptane. These results are promising and highlight the use of the FS containing PR10110 lipases for the first time in biocatalytic processes.

Immobilization and bioimprinting strategies to enhance the performance in organic medium of the metagenomic lipase LipC12.

The present work evaluates the immobilization of LipC12 on different supports in tandem with bioimprinting technique, in order to improve its activity and stability in organic medium. Oleic acid was selected as the bioimprinting molecule. The immobilized LipC12 was applied in the synthesis of pentyl oleate by esterification reaction and in the production of fatty acids, mono, and diglycerides via hydrolysis of triacylglycerols, in n-heptane reaction media. For all immobilized lipase preparations, an increase in the conversion of oleic acid to pentyl oleate was observed when immobilization in tandem with bioimprinting treatment was carried out versus immobilization without bioimprinting. The highest conversions were achieved using LipC12 immobilized on hydrophobic supports. The reuse potential of the immobilized preparations was evaluated. The preparations were used in eight successive cycles of esterification reactions and the best results were obtained for LipC12 immobilized on Immobead 150 and chitosan. The activity for the hydrolysis of soybean oil was improved by bioimprinting treatment only for LipC12 immobilized on commercial polypropylene and Accurel MP-1000. LipC12 immobilized on hydrophilic supports or on Immobead150 could be used to hydrolyze tricaprylin to obtain diglycerides with a high proportion of 1,2-diglycerides in reaction times as short as 30 min.

Potential of time-stepping stochastic models as tools for guiding the design and operation of processes for the enzymatic hydrolysis of polysaccharides - A review.

Processes for the enzymatic hydrolysis of polysaccharides in biorefineries are becoming increasingly important. The complex network of reactions involved in polysaccharide hydrolysis can be described by stochastic models that advance in steps of time. Such models have the potential to be important tools for guiding process design and operation, and several have been developed over the last two decades. We evaluate these models. Many of the current stochastic models for the hydrolysis of colloidal polysaccharides use empirical parameters that have no recognized biological meaning. Only one model uses classical parameters of enzyme kinetics, namely specificity constants and saturation constants. Recent stochastic models for the hydrolysis of insoluble cellulose give valuable insights into the molecular-level phenomenon that limit hydrolysis rates. We conclude that, if stochastic models of enzymatic polysaccharide hydrolysis are to become widely used tools for guiding process development, then further improvements are required.

Performing under pressure: esterification activity of dry fermented solids in subcritical and supercritical CO2.

OBJECTIVE: Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the "dry fermented solids" (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical CO2 at 40 °C. RESULTS: Compared to a control without CO2 at atmospheric pressure, subcritical CO2 at 30 bar improved esterification activity 1.2-fold. Higher pressures, including supercritical pressures up to 150 bar, reduced activity to less than 80% of the control. At 30 bar, the esterification activity was improved a further 1.8-fold with the addition of 9% water (i.e. 9 g water per 100 g oleic acid) to the reaction medium. CONCLUSION: A subcritical CO2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.

Immobilization of Pseudomonas cepacia lipase on layered double hydroxide of Zn/Al-Cl for kinetic resolution of rac-1-phenylethanol.

Layered double hydroxides (LDHs) are cheap materials suitable for immobilization of enzymes. In this study, we prepared Zn/Al-Cl LDHs with different Zn:Al molar ratios for immobilization of the lipase from Pseudomonas cepacia. The best values for activity retention (188%), immobilization efficiency (96%) and hydrolytic activity in organic medium (279 U g-1) were obtained with a molar ratio of Zn:Al of 4:1, a protein loading of 162 mg g-1 and Tris-HCl buffer (10 mmol L-1, pH 7.5) as the solvent for preparing the lipase solution. The immobilized lipase keeps its activity when stored at 4 °C during 30 days. The immobilized lipase gave a conversion of 50% in 1 h for the kinetic resolution of the alcohol rac-1-phenylethanol, with both ees and eep higher than 99% and E higher than 200. In the reutilization study, 30 successive 1-h kinetic resolutions were done with the same batch of immobilized enzyme. For all 30 resolutions, 50% conversion was maintained, with ees and eep higher than 99% and E higher than 200. These are promising results that lay the basis for further studies of immobilization of lipases onto LDHs for applications in organic media.

Structure solution and analyses of the first true lipase obtained from metagenomics indicate potential for increased thermostability.

Metagenomics is a modern approach to discovery of new enzymes with novel properties. This article reports the structure of a new lipase, belonging to family I.1, obtained by means of metagenomics. Its structure presents a fold typical of α/ß hydrolases, with the lid in closed conformation. The protein was previously shown to present high thermostability and to be stable in aqueous solutions of polar organic solvents at high concentrations [30% (V/V)]. Molecular dynamics studies showed that the protein maintains its structure well in organic solvents. They also suggested that its thermostability might be enhanced if it were mutated to present a disulfide bond similar to that typically found in lipase family I.2. These findings identify this lipase as a good candidate for further improvement through protein engineering.

Biochemical characterization and application of a new lipase and its cognate foldase obtained from a metagenomic library derived from fat-contaminated soil.

LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40 °C and pH 6.5. Under these conditions, the highest activity was against tributyrin (1650 U mg-1), but it also had high activity against olive oil (862 U mg-1). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24 h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24 h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5 h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)­1­phenylethyl acetate for the kinetic resolution of (R,S)­1­phenyl­1­ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.

Genome sequencing of Burkholderia contaminans LTEB11 reveals a lipolytic arsenal of biotechnological interest.

Burkholderia contaminans LTEB11 is a Gram-negative betaproteobacterium isolated as a contaminant of a culture in mineral medium supplemented with vegetable oil. Here, we report the genome sequence of B. contaminans LTEB11, identifying and analyzing the genes involved in its lipolytic machinery and in the production of other biotechnological products.

Design and Operation of a Pilot-Scale Packed-Bed Bioreactor for the Production of Enzymes by Solid-State Fermentation.

In this review, we describe our experience in building a pilot-scale packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent mixing, and the use of this bioreactor to produce pectinases and lipases by filamentous fungi. We show that, at pilot scale, special attention must be given to several aspects that are not usually problematic when one works with laboratory-scale SSF bioreactors. For example, it can be a challenge to produce large amounts of inoculum if the fungus does not sporulate well. Likewise, at larger scales, the air preparation system needs as much attention as the bioreactor itself. Sampling can also be problematic if one wishes to avoid disrupting the bed structure. In the fermentations carried out in the pilot bioreactor, when the substrate bed contained predominantly wheat bran, the bed shrank away from the walls, providing preferential flow paths for the air and necessitating agitation of the bed. These problems were avoided by using beds with approximately 50% of sugarcane bagasse. We also show how a mathematical model that describes heat and water transfer in the bed can be a useful tool in developing appropriate control schemes. Graphical Abstract.

Fermented Solids and Their Application in the Production of Organic Compounds of Biotechnological Interest.

We review the application of dry fermented solids (DFS) containing naturally immobilized enzymes as catalysts in synthesis and in hydrolysis reactions. The most studied application is the use of DFS containing lipases in the synthesis of biodiesel esters, by transesterification of oils or by esterification of fatty acids with short-chain alcohols in solvent-free reaction media. Other applications of DFS that have been studied include the use of DFS containing pectinases to liberate D-galacturonic acid from pectin and the production of high-value compounds by DFS containing lipases, such as the synthesis of sugar esters and the production of pure enantiomers by resolution of racemic mixtures. To date, studies are limited to proof of concept, and there are still many challenges to be faced in the development of industrial-scale processes using DFS as catalysts. A key challenge is the relatively low activity of DFS compared to commercial enzyme preparations. Attention needs to be given to scale up, not only of the bioreactor for the application of the DFS but also for the production of the fermented solids. There is also a need for economic feasibility studies to determine whether the production of DFS and their use as catalysts can be competitive at industrial scale. Graphical Abstract.

Tailoring recombinant lipases: keeping the His-tag favors esterification reactions, removing it favors hydrolysis reactions.

We determined the effect of the His-tag on the structure, activity, stability and immobilization of LipC12, a highly active lipase from a metagenomic library. We purified LipC12 with a N-terminal His-tag and then removed the tag using tobacco etch virus (TEV) protease. Circular dichroism analysis showed that the overall structure of LipC12 was largely unaffected by His-tag removal. The specific hydrolytic activities against natural and artificial substrates were significantly increased by the removal of the His-tag. On the other hand, His-tagged LipC12 was significantly more active and stable in the presence of polar organic solvents than untagged LipC12. The immobilization efficiency on Immobead 150 was 100% for both forms of LipC12 and protein desorption studies confirmed that the His-tag does not participate in the covalent binding of the enzyme. In the case of immobilized LipC12, the His-tag negatively influenced the hydrolytic activity, as it had for the free lipase, however, it positively influenced the esterification activity. These results raise the possibility of tailoring recombinant lipases for different applications, where the His-tag may be retained or removed, as appropriate for the desired activity.

Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg-1) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n­hexane with an activity of 4 U g-1, maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis.

Optimization of biodiesel synthesis by esterification using a fermented solid produced by Rhizopus microsporus on sugarcane bagasse.

A fermented solid containing lipases was produced by solid-state fermentation of Rhizopus microsporus on sugarcane bagasse enriched with urea, soybean oil, and a mineral solution. The dry fermented solid produced using R. microsporus (RMFS) was used to catalyze the synthesis of alkyl-esters by esterification in a solvent-free system containing ethanol and oleic acid (as a model system) or a mixture of fatty acids obtained from the physical hydrolysis of soybean soapstock acid oil (FA-SSAO) in subcritical water. The conversions were 93.5 and 84.1%, for oleic acid and FA-SSAO, respectively, at 48 h and 40 °C, at a molar ratio (MR) of ethanol to fatty acid of 5:1. A further increase in the MR to 10:1 improved the production of ethylic-esters, giving conversions at 48 h of 98 and 86% for oleic acid and FA-SSAO, respectively. The results obtained in this work foster further studies on scaling-up of an environmentally friendly process to produce biofuels.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candidarugosa lipase, metagenomic lipase, ß-galactosidase and ß-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae.

BACKGROUND: Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize D-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp. RESULTS: In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for D-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described D-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized D-galacturonic acid in a medium containing D-fructose as co-substrate. CONCLUSION: This work is the first demonstration of the expression of a functional heterologous pathway for D-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.